Modified Project Summary/Abstract Section This project will be continued at Princeton University in the Princeton Neuroscience Institute. Early life stress (ELS) is one of the strongest lifetime risk factors for developing depression and other psychiatric disorders, particularly after facing additional stressful events in adulthood. However, relatively little is known about how the developing brain encodes experience of ELS to increase sensitivity to additional stress. To address this, we have established a translationally relevant ?two-hit? mouse model of early life and adult stress, wherein stress during a specific postnatal sensitive window increases the likelihood that stress in adulthood will lead to depressive-like behaviors. This proposal will examine whether changes in transcription, epigenetic regulation, and cellular microcircuit reactivation are induced by ELS experience, and whether these changes directly contribute to stress-sensitized depression-like behaviors. In Aims 1 and 3, we will investigate changes in transcription and ?priming? of the epigenetic landscape using a variety of cutting-edge techniques including RNA sequencing, chromatin immunoprecipitation, and ATAC-sequencing. In Aim 2, we will ask whether cells in mesocorticolimbic reward-related brain regions activated by ELS are reactivated by adult stress, using activity dependent transgenic mice (ArcCreERT2). In Aim 3 we will then combine these approaches using a novel transgenic cross (ArcCreERT2 x R26-CAGLSL- Sun1-sfGFP-myc) to examine, for the first time, changes in epigenetic priming specifically within nuclei isolated from ELS-activated cells. We will then use pharmacogenetic inhibition to assess the necessity of cells initially activated by ELS for subsequent stress sensitization and depression-like behavior. In addition, we will use CRISPR constructs to knock out Setd7, an enzyme that establishes epigenetic priming by H3K4me1 deposition. This will allow us to test the functional relevance of this mark for the stress sensitization effects of ELS. In sum, the research proposed in this Pathway to Independence Award will reveal both separate and potentially interactive molecular and cellular mechanisms of long-lasting ELS-induced stress sensitization and enhanced vulnerability to depression and psychiatric disease.